Atrophic rhinitis (hereinafter also referred to as "AR") is a swine chronic respiratory disease which has extremely potent infectivity with primary symptoms being turbinate atrophy, hypoplasia, epistaxis, twisting of the snout and induction of other respiratory diseases, and the like. This disease is known to be caused by a toxigenic Bordetella bronchiseptica (hereinafter also referred to as "Bb") and a toxigenic Pasteurella multocida (hereinafter also referred to as "Pm") which infect at the mucosa of an upper respiratory tract. Bb possesses hemagglutinin and pili as an adherence factor and thus easily attaches to the nasal mucosa while Pm does not have such an adherence factor and hence hardly colonizes by itself. Accordingly, for the establishment of Pm infection, a predisposing factor causing a lesion at the nasal mucosa is necessary, said factor being typically Bb. Pathological factors involved in the cause of lesion in the nasal mucosa are known to be a tracheal cytotoxin and a dermonecrotic toxin. In an experimental infection, a pig is infected with Bb, and several days later, followed by intranasal infection with Pm, to cause a severe AR as observed in a field farm.
Bordetella includes B. pertussis, B. parapertussis, B. bronchiseptica, B. avium, and the like, which produce a variety of biologically active substances. Bb dermonecrotic toxin (hereinafter also referred to as "BbT") is one of such substances and is produced by any Bordetella. BbT having biological activities such as a hemorrhagic necrotic activity, an activity to cause turbinate atrophy, an activity to cause hypoplasia, a lethal activity, a vasoconstrictive activity in the smooth muscle, an activity to cause interruption in a local blood circulation, an activity to cause splenoatrophy, an activity to inhibit an antibody production, and the like was found to play an important role in Bordetellosis. Furthermore, it was found that BbT also plays an important role in the formation of pneumonic focus in piglets (Roop II, R. M. et al., Infect. Immun., 55, p.217-222 (1987)).
On the other hand, a part of Pasteurella multocida or a part of atypical Pasteurella produce Pm dermonecrotic toxin (hereinafter also referred to as "PmT"). PmT having biological activities such as a hemorrhagic necrotic activity, an activity to cause turbinate atrophy, an activity to cause hypoplasia, a lethal activity, an activity to cause interruption in a local blood circulation, an activity to cause splenoatrophy, and the like has been found to play an important role in the onset of disease by the toxigenic Pm. Furthermore, it was found that PmT is closely related to the formation of pneumonic focus in pigs by the toxigenic Pm (Iwatsu, S. and Sawada, T.; Jpn. J. Vet. Sci., 50, p.1200-1206 (1988)). Although both BbT and PmT are not secreted into a culture medium but accumulate within bacterial cells, they are released out of the bacterial cells into a culture supernatant due to bacteriolysis after a long term culture. In the in vivo process, dermonecrotic toxin released out of the bacterial cells due to bacteriolysis affect the living body.
Under the above-mentioned circumstances, the role of dermonecrotic toxin in protection in the living body still remains unclear, and for elucidating the mechanism of the protection, there is a need to develop a process for a large scale preparation of dermonecrotic toxin and to develop a toxoid of dermonecrotic toxin for use in prophylaxis of the disease. However, it has been difficult to purify dermonecrotic toxin due to its properties, i.e. unstability to heat, self-agglutination easily caused as purification processes proceed, formation of a complex with bacterial cell-derived substances such as lipids, acid proteins, hydrophobic substances, etc. Furthermore, since BbT and PmT do not immunologically cross-react to each other in spite of their similarity in biological activities, it has been earnestly desired to develop a BbT-PmT toxoid mixture which is safe and effective from the viewpoint of veterinary and pig breeding industry.
A hitherto known process for partially purifying dermonecrotic toxin from a bacterial dermonecrotic toxin-containing solution, especially from a Bordetella bacterial cell extract, includes a process using a ion exchange chromatography (Kume, K. et al., Infect. Immun., 52(4), p.370-377 (1986)). However, this process disadvantageously shows poor reproducibility. A process for partially purifying dermonecrotic toxin is also known which utilizes dialysis, a sucrose-gradient ultracentrifugation and a gel filtration chromatography (Endoh, M. et al., Microbiol. Immunol., 30, (7), p.659-673 (1986)). However, this process also shows disadvantageously poor reproducibility, and in addition, does not provide a sufficient degree of purification in spite of many steps.
Another example includes a process utilizing treatment with a nucleic acid-removing agent, an anion exchange chromatography and a hydroxyapatite adsorption chromatography (Horiguchi, Y. et al., Microbiol. Pathogen., 6, p.361-368 (1989)). However, this process is also disadvantageous in that it requires a costly reagent, a nucleic acid-removing agent, and involves many steps. There is also a process utilizing a nucleic acid-removing agent, a dialysis and a cation exchange chromatography (Horiguchi, Y. et al., FEMS Microbiol. Letters, 66, p.39-44 (1990)). Although this process shows high reproducibility and can provide dermonecrotic toxin with comparatively high purity, it also disadvantageously requires a costly reagent as in the above process.
Still another example is a process using a salting-out and a dye ligand affinity chromatography (Zhang Y. L. and Sekura R. D., Infect. Immun., 59(10), p.3754-3759 (1991)). Although this process shows a high reproducibility, it disadvantageously provides an insufficient degree of purification.
Inactivated AR vaccines reported so far includes three types, i.e. an inactivated Bb plain vaccine, an inactivated Pm plain vaccine and a Bb/Pm combined inactivated vaccine, each having been studied or put into practical use.
A Bb inactivated vaccine includes one comprising killed bacteria inactivated with formalin, glutaraldehyde, etc. supplemented with aluminum gel or an oil adjuvant (Goodnow, R. A., Vet. Med. Small Anim. Clin., 72, p.1210-1212 (1977); Nakase, Y. et al., Proc. Int. Pig Vet. Soc. Congr., 8, (1976); Giles, C. J. and Smith, I. M., Vet. Bull.(London), 53, p.327-338 (1983)). It was also found that a 68 kDa protein, which is present on the outer membrane of Bb and has an adenylate cyclase activity, exhibits a prophylactic activity against atrophic rhinitis (Montaraz J. A. et al., Infect. Immun., 47,, p.744-755 (1985)). A component vaccine comprising a Bb-produced fibrous hemagglutinin as a primary component is also known (Hansen, G. R. et al.; In Atrophic rhinitis in pigs (Ed. Peterson, K. B. and Nielsen, N. C.) Communication of the European Communities, Luxembourg, p.89-97 (1983), and Ohgitani, T. et al., Vaccine, 9, p.653-658 (1991)). There is also reported that a vaccine supplemented with dermonecrotic toxin for providing an antitoxin is not efficacious (Soderlind, O. and Bergstrom, G., Proc. Int. Pig Vet. Sci. Congr., p.175 (1984); Marel, C. M. von der. et al., Proc. Int. Pig Vet. Sci. Congr., p.170 (1984)).
On the other hand, an inactivated Pm plain vaccine includes Pm toxoid (Pedersen, K. B. and Barfod, K., Nord. Vet. Med., 31, p.293-302 (1982); Foged, N. T. et al., Vet. Rec., 125, p.7-11 (1989)), which has been studied or put into practical use.
A Bb/Pm combined inactivated vaccine includes (1) a mixture of killed Bb with killed PmT-producing PmD type bacteria, the mixture which is further supplemented with PmT non-producing bacteria, or a mixture of Bb and PmT toxoid (Barfod, K. and Pedersen, K. B., Nord. Vet. Med., 36, p.337-345 (1984); Baalsrud, K. J., Acta Vet. Scand., 28, p.305-311 (1987)); de Jong, M. F. et al., Vet. Quart., 9, p.49-59 (1987); Kobisch, M. and Pennings, A., Vet. Rec., 124, p.57-61 (1989)), for the purpose of prophylaxis of AR alone; (2) a mixture of killed Bb with killed PmD type PmT-producing bacteria and killed PmA type bacteria (Ostle, A. G. et al., Vet. Med., p.772-775 (1986)), for the purpose of prophylaxis of AR and pneumonia; and (3) a mixture of killed Bb with killed PmA type bacteria, killed Erysipelothrix insidiosa and PmT toxoid (Jayappa, H. et al., Int. Pig Vet. Soc. Congr., p.44 (1988)), for the purpose of prophylaxis of other diseases as well as AR and pneumonia, any of (1) to (3) having been put into practical use.
Since BbT plays an important role in the turbinate atrophy and the pneumonic focus formation by Bb infection, Zoop II, R.M. et al. has pointed out a need of BbT toxoid (Infect. Immun., 55, p.217-222 (1987)). However, they never refer to a combination of BbT and PmT toxoids. Chanter, N. et al. has proposed that a combination of PmT toxoid with the important toxin of Bb is more effective since the lesion in the swine nasal mucosa induced by cooperative activity of BbT and PmT provides environments favorable to Bb and Pm propagation (Res. Vet. Sci., 47, p. 48-53 (1989)).
However, a production of a BbT neutralizing antibody has not yet been successfully enhanced with a vaccine comprising a fraction having a BbT activity (Soderlind, O. and Bergstrom, G., Proc. Int. Pig Vet. Sci. Congr., p. 175 (1984); Marel, C. M. von der. et al., Proc. Int. Pig Vet. Sci. Congr., p.170 (1984)). On the other hand, Nakai et al. revealed that a production of a neutralizing antibody is enhanced in pigs and mice with a highly purified BbT toxoid (The 111th Japan Veterinary Society, lecture abstract, p.183 (1991)). However, they also never refer to a combination of said BbT toxoid and PmT toxoid.